Pathogen Trypanosoma Cruzi Research Articles

Higher expression of proline dehydrogenase altered mitochondrial function and increased Trypanosoma cruzi differentiation in vitro and in the insect vector.

The pathogenic protist Trypanosoma cruzi uses kissing bugs as invertebrate hosts that vectorize the infection among mammals. This parasite oxidizes proline to glutamate through two enzymatic steps and one nonenzymatic step. In insect vectors, T. cruzi differentiates from a noninfective replicating form to nonproliferative infective forms. Proline sustains this differentiation, but to date, a link between proline metabolism and differentiation has not been established. In T. cruzi, the enzymatic steps of the proline-glutamate oxidation pathway are catalyzed exclusively by the mitochondrial enzymes proline dehydrogenase [TcPRODH, EC: 1.5.5.2] and Δ1-pyrroline-5-carboxylate dehydrogenase [TcP5CDH, EC: 1.2.1.88]. Both enzymatic steps produce reducing equivalents that are able to directly feed the mitochondrial electron transport chain (ETC) and thus produce ATP. In this study, we demonstrate the contribution of each enzyme of the proline-glutamate pathway to ATP production. In addition, we show that parasites overexpressing these enzymes produce increased levels of H2O2, but only those overexpressing TcP5CDH produce increased levels of superoxide anion. We show that parasites overexpressing TcPRODH, but not parasites overexpressing TcP5CDH, exhibit a higher rate of differentiation into metacyclic trypomastigotes in vitro. Finally, insect hosts infected with parasites overexpressing TcPRODH showed a diminished parasitic load but a higher percent of metacyclic trypomastigotes, when compared with controls. Our data show that parasites overexpressing both, PRODH and P5CDH had increased mitochondrial functions that orchestrated different oxygen signaling, resulting in different outcomes in relation to the efficiency of parasitic differentiation in the invertebrate host.

Novel Therapeutic Options for Chagas Disease Based on Bioactive Compounds from Algae, Bacteria and Fungi Species.

Chagas Disease, also known as American trypanosomiasis, is a Neglected Tropical Disease that affects around seven million people, especially in Latin America. Noteworthy, there has been an increase in the numbers of case reports in non-endemic areas, such as North America, Europe, Japan, and Australia. The disease is a vector-borne disease caused by the pathogen Trypanosoma cruzi being transmitted by infected bugs. It is known that about forty percent of infected patients develop cardiac, digestive, or neurological alterations. There are only two drugs currently used for treatment, benznidazole and nifurtimox. However, both therapeutic regimens present several limitations, such as toxicity, mutagenicity and low efficiency during the chronic phase. Some reports in the literature point to the occurrence of parasite resistance. To overcome these limitations, the bioprospection of novel molecules as alternatives is one of the major goals to improve therapeutic success in this chronic disease. Bioprospecting active metabolites from natural resources might bring new hopes for disease control and parasite elimination. Here we summarize the most recent advances to identify and test Algae, Bacteria and Fungi-derived bioactive compounds with trypanocidal activity using experimental models, in vitro testing and in silico approaches.

Drug Target Validation of the Protein Kinase AEK1, Essential for Proliferation, Host Cell Invasion, and Intracellular Replication of the Human Pathogen Trypanosoma cruzi.

ABSTRACTProtein phosphorylation is involved in several key biological roles in the complex life cycle of Trypanosoma cruzi, the etiological agent of Chagas disease, and protein kinases are potential drug targets. Here, we report that the AGC essential kinase 1 (TcAEK1) exhibits a cytosolic localization and a higher level of expression in the replicative stages of the parasite. A CRISPR/Cas9 editing technique was used to generate ATP analog-sensitive TcAEK1 gatekeeper residue mutants that were selectively and acutely inhibited by bumped kinase inhibitors (BKIs). Analysis of a single allele deletion cell line (TcAEK1-SKO), and gatekeeper mutants upon treatment with inhibitor, showed that epimastigote forms exhibited a severe defect in cytokinesis. Moreover, we also demonstrated that TcAEK1 is essential for epimastigote proliferation, trypomastigote host cell invasion, and amastigote replication. We suggest that TcAEK1 is a pleiotropic player involved in cytokinesis regulation in T. cruzi and thus validate TcAEK1 as a drug target for further exploration. The gene editing strategy we applied to construct the ATP analog-sensitive enzyme could be appropriate for the study of other proteins of the T. cruzi kinome.IMPORTANCE Chagas disease affects 6 to 7 million people in the Americas, and its treatment has been limited to drugs with relatively high toxicity and low efficacy in the chronic phase of the infection. New validated targets are needed to combat this disease. In this work, we report the chemical and genetic validation of the protein kinase AEK1, which is essential for cytokinesis and infectivity, using a novel gene editing strategy.

Chagas Disease: Detection of Trypanosoma cruzi by a New, High-Specific Real Time PCR.

Background: Chagas disease (CD) is a major burden in Latin America, expanding also to non-endemic countries. A gold standard to detect the CD causing pathogen Trypanosoma cruzi is currently not available. Existing real time polymerase chain reactions (RT-PCRs) lack sensitivity and/or specificity. We present a new, highly specific RT-PCR for the diagnosis and monitoring of CD. Material and Methods: We analyzed 352 serum samples from Indigenous people living in high endemic CD areas of Colombia using three leading RT-PCRs (k-DNA-, TCZ-, 18S rRNA-PCR), the newly developed one (NDO-PCR), a Rapid Test/enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence. Eighty-seven PCR-products were verified by sequence analysis after plasmid vector preparation. Results: The NDO-PCR showed the highest sensitivity (92.3%), specificity (100%), and accuracy (94.3%) for T. cruzi detection in the 87 sequenced samples. Sensitivities and specificities of the kDNA-PCR were 89.2%/22.7%, 20.5%/100% for TCZ-PCR, and 1.5%/100% for the 18S rRNA-PCR. The kDNA-PCR revealed a 77.3% false positive rate, mostly due to cross-reactions with T. rangeli (NDO-PCR 0%). TCZ- and 18S rRNA-PCR showed a false negative rate of 79.5% and 98.5% (NDO-PCR 7.7%), respectively. Conclusions: The NDO-PCR demonstrated the highest specificity, sensitivity, and accuracy compared to leading PCRs. Together with serologic tests, it can be considered as a reliable tool for CD detection and can improve CD management significantly.

Glucose-6-Phosphate Dehydrogenase from the Human Pathogen Trypanosoma cruzi Evolved Unique Structural Features to Support Efficient Product Formation

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer–dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

Molecular and biological characterization of a highly pathogenic Trypanosoma cruzi strain isolated from a patient with congenital infection

Although many Trypanosoma cruzi (T. cruzi) strains isolated from a wide range of hosts have been characterized, there is a lack of information about biological features from vertically transmitted strains.We describe the molecular and biological characteristics of the T. cruzi VD strain isolated from a congenital Chagas disease patient.The VD strain was typified as DTU TcVI; in vitro sensitivity to nifurtimox (NFX) and beznidazole (BZ) were 2.88 μM and 6.19 μM respectively, while inhibitory concentrations for intracellular amastigotes were 0.24 μM for BZ, and 0.66 μM for NFX. Biological behavior of VD strain was studied in a mouse model of acute infection, resulting in high levels of parasitemia and mortality with a rapid clearence of bloodstream trypomastigotes when treated with BZ or NFX, preventing mortality and reducing parasitic load and intensity of inflammatory infiltrate in skeletal and cardiac muscle. Treatment-induced parasitological cure, evaluated after immunossupression were 41% and 35% for BZ and NFX treatment respectively, suggesting a partial response to these drugs in elimination of parasite burden.This exhaustive characterization of this T. cruzi strain provides the basis for inclusion of this strain in a panel of reference strains for drug screening and adds a new valuable tool for the study of experimental T. cruzi infection.

Trypanosoma cruzi Infection Prevalence and Bloodmeal Analysis in Triatomine Vectors of Chagas Disease From Rural Peridomestic Locations in Texas, 2013-2014.

Protozoan pathogen Trypanosoma cruzi (Chagas, 1909) is the etiologic agent of Chagas disease, which affects millions of people in Latin America. Recently, the disease has been gaining attention in Texas and the southern United States. Transmission cycle of the parasite involves alternating infection between insect vectors and vertebrate hosts (including humans, wildlife, and domestic animals). To evaluate vector T. cruzi parasite burden and feeding patterns, we tested triatomine vectors from 23 central, southern, and northeastern counties of Texas. Out of the 68 submitted specimens, the majority were genetically identified as Triatoma gerstaeckeri (Stal, 1859), with a few samples of Triatoma sanguisuga (LeConte, 1855), Triatoma lecticularia (Stal, 1859), Triatoma rubida (Uhler, 1894), and Triatoma protracta woodi (Usinger, 1939). We found almost two-thirds of the submitted insects were polymerase chain reaction-positive for T. cruzi Bloodmeal sources were determined for most of the insects, and 16 different species of mammals were identified as hosts. The most prevalent type of bloodmeal was human, with over half of these insects found to be positive for T. cruzi High infection rate of the triatomine vectors combined with high incidence of feeding on humans highlight the importance of Chagas disease surveillance in Texas. With our previous findings of autochthonous transmission of Chagas disease, urgent measures are needed to increase public awareness, vector control in and around homes, and Chagas screening of residents who present with a history of a triatomine exposure.

Binding Mode and Selectivity of Steroids towards Glucose-6-phosphate Dehydrogenase from the Pathogen Trypanosoma cruzi.

Glucose-6-phosphate dehydrogenase (G6PDH) plays a housekeeping role in cell metabolism by generating reducing power (NADPH) and fueling the production of nucleotide precursors (ribose-5-phosphate). Based on its indispensability for pathogenic parasites from the genus Trypanosoma, G6PDH is considered a drug target candidate. Several steroid-like scaffolds were previously reported to target the activity of G6PDH. Epiandrosterone (EA) is an uncompetitive inhibitor of trypanosomal G6PDH for which its binding site to the enzyme remains unknown. Molecular simulation studies with the structure of Trypanosoma cruzi G6PDH revealed that EA binds in a pocket close to the G6P binding-site and protrudes into the active site blocking the interaction between substrates and hence catalysis. Site directed mutagenesis revealed the important steroid-stabilizing effect of residues (L80, K83 and K84) located on helix α-1 of T. cruzi G6PDH. The higher affinity and potency of 16α-Br EA by T. cruzi G6PDH is explained by the formation of a halogen bond with the hydrogen from the terminal amide of the NADP+-nicotinamide. At variance with the human enzyme, the inclusion of a 21-hydroxypregnane-20-one moiety to a 3β-substituted steroid is detrimental for T. cruzi G6PDH inhibition. The species-specificity of certain steroid derivatives towards the parasite G6PDH and the corresponding biochemically validated binding models disclosed in this work may prove valuable for the development of selective inhibitors against the pathogen’s enzyme.

Quantitative enzymatic production of sialylated galactooligosaccharides with an engineered sialidase from Trypanosoma rangeli

Sialylated galactooligosaccharides (GOS) represent a potential infant formula ingredient, which is believed to contribute with a combination of the beneficial properties of the prebiotic GOS as well as of sialylated human milk oligosaccharides. Sialylated GOS do not exist in natural milk, but can be produced from κ(kappa)-casein glycomacropeptide (CGMP), a sialylated side stream component from cheese-making, by sialidase-catalyzed transsialylation. Using a rationally designed mutant of the sialidase from Trypanosoma rangeli, Tr13, with enhanced transsialylation activity, six different GOS preparations with a varying degree of polymerization (DP) were effectively sialylated with molar yields of 20–30% on the CGMP sialyl in batch reactions. The rate of sialylation of the individual DPs was largely dependent on the DP distribution in each GOS preparation, and Tr13 catalysis did not discriminate against large GOS molecules, providing the novelty point that GOS molecules are sialylated independently of their size by Tr13. Using CGMP, GOS, and Tr13, the production of gram-scale quantities of sialyl-GOS was achieved in 20L volume reactions. Compared to the benchmark transsialidase from pathogenic Trypanosoma cruzi, the Tr13 was significantly more thermostable. By employing an enzymatic membrane reactor, Tr13 could be recycled and after seven consecutive 1-h reaction cycles, the biocatalytic productivity of the enzyme was increased 7-fold compared to the batch reaction. Assuming that the enzyme may be specific for α-2,3-bound sialyl moieties only, and that only 50% of sialyl linkages in CGMP are α-2,3-linked, the molar yield of sialyl-GOS on the available α-2,3-bound sialyl moieties in CGMP reached 80% in the enzymatic membrane reactor system.

Cruzipain: An Update on its Potential as Chemotherapy Target against the Human Pathogen Trypanosoma cruzi.

Chagas' disease is one of the most impactful and prevalent neglected tropical diseases in the Americas, specially affecting the poor and underdeveloped areas in Latin America. Aggravating this scenario, the medicines used in the current chemotherapy are old, toxic and present a low efficacy to treat the chronic stage of this disease. In addition, resistant strains of Trypanosoma cruzi, the etiological agent, are frequently reported. So, there is an imperative requirement for novel chemotherapeutic options to treat this debilitating disease. In this context, peptidases have emerged as potential targets and, consequently, proteolytic inhibitors have confirmed to be valuable drugs against several human pathologies. In this line of thinking, T. cruzi produces a major multifunctional cysteine peptidase, named cruzipain, which directly and/or indirectly orchestrates several physiological and pathological processes, which culminate in a successful parasitic infection. Taken together, these findings point out that cruzipain is one of the most important targets for driving a chemotherapy approach against the human pathogen T. cruzi. The present review summarizes some of the recent advances and failures in this area, with particular emphasis on recently published studies.

The GTPase TcRjl of the human pathogen Trypanosoma cruzi is involved in the cell growth and differentiation

The protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas Disease, undergoes through a complex life cycle where rounds of cell division and differentiation occur initially in the gut of triatominae vectors and, after transmission, inside of infected cells in vertebrate hosts. Members of the Ras superfamily of GTPases are molecular switches which play pivotal regulatory functions in cell growth and differentiation. We have previously described a novel GTPase in T. cruzi, TcRjl, which belongs to the RJL family of Ras-related GTP binding proteins. Here we show that most of TcRjl protein is found bound to GTP nucleotides and may be locked in this stage. In addition, we show that TcRjl is located close to the kinetoplast, in a region corresponding possibly to flagellar pocket of the parasite and the expression of a dominant-negative TcRjl construct (TcRjlS37N) displays a significative growth phenotype in reduced serum medium. Remarkably, overexpression of TcRjl inhibits differentiation of epimastigotes to trypomastigote forms and promotes the accumulation of intermediate differentiation stages. Our data suggest that TcRjl might play a role in the control of the parasite growth and differentiation.

Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development.

Galactose‐Phosphonates as Mimetics of the Sialyltransfer by Trypanosomal Sialidases

In an attempt to find competitive inhibitors of the trans‐sialidase of the pathogen Trypanosoma cruzi, we have synthesized conjugates of carbocyclic sialylmimetics (e.g., cyclohexenephosphonates) and galactose derivatives. A trans‐sialidase inhibition assay revealed an interesting preference for ethylidene‐spacered conjugates involving the 3‐position of the sugar.

Inhibition of host connective tissue growth factor expression: a novel Trypanosoma cruzi‐mediated response

Connective tissue growth factor (CTGF) is a secreted cytokine that plays a fundamental role in the development of tissue fibrosis by mediating many of the profibrotic effects of TGF-beta. We present the novel finding that the intracellular pathogen Trypanosoma cruzi elicits immediate and sustained repression of basal CTGF expression in dermal fibroblasts, followed by down-regulation of the extracellular matrix proteins, fibronectin, and collagen I alpha1. To address mechanisms underlying this response, the major CTGF-regulating pathways were investigated. We report that both T. cruzi trypomastigotes and secreted parasite factor(s) antagonize TGF-beta-dependent induction of CTGF in fibroblasts. Of the TGF-beta-dependent signaling pathways required for CTGF expression, we demonstrate that T. cruzi interferes with cellular Erk1/2 phosphorylation but not Smad3 signaling. While increased stimulation of Erk phosphorylation alone was insufficient to override the parasite-mediated repression of CTGF, stimulation of fibroblasts with increased concentrations of TGF-beta, which activates both Smad3 and Erk1/2, completely abrogated this inhibition. Together with the finding that T. cruzi-mediated down-regulation of CTGF expression requires de novo host cell protein synthesis, our data indicate that the unique ability of T. cruzi to interfere with the host fibrogenic response is a complex process requiring input from multiple host cell signaling pathways.

Crystal structure of Trypanosoma cruzi trypanothione reductase in complex with trypanothione, and the structure-based discovery of new natural product inhibitors.

Trypanothione reductase (TR) helps to maintain an intracellular reducing environment in trypanosomatids, a group of protozoan parasites that afflict humans and livestock in tropical areas. This protective function is achieved via reduction of polyamine-glutathione conjugates, in particular trypanothione. TR has been validated as a chemotherapeutic target by molecular genetics methods. To assist the development of new therapeutics, we have characterised the structure of TR from the pathogen Trypanosoma cruzi complexed with the substrate trypanothione and have used the structure to guide database searches and molecular modelling studies. The TR-trypanothione-disulfide structure has been determined to 2.4 A resolution. The chemical interactions involved in enzyme recognition and binding of substrate can be inferred from this structure. Comparisons with the related mammalian enzyme, glutathione reductase, explain why each enzyme is so specific for its own substrate. A CH***O hydrogen bond can occur between the active-site histidine and a carbonyl of the substrate. This interaction contributes to enzyme specificity and mechanism by producing an electronic induced fit when substrate binds. Database searches and molecular modelling using the substrate as a template and the active site as receptor have identified a class of cyclic-polyamine natural products that are novel TR inhibitors. The structure of the TR-trypanothione enzyme-substrate complex provides details of a potentially valuable drug target. This information has helped to identify a new class of enzyme inhibitors as novel lead compounds worthy of further development in the search for improved medicines to treat a range of parasitic infections.

Hydrophobic attachment of Trypanosoma cruzi to a superficial layer of the rectal cuticle in the bug Triatoma infestans.

In the vector Triatoma infestans the human pathogenic flagellate Trypanosoma cruzi colonizes mainly the rectum. The rectal cuticle and associated trypanosomes were examined by electron microscopy. The cuticle of the rectum consisted of a superficial wax layer, which was not retained by conventional preparation, an outer and inner epicuticle, and a procuticle. Epimastigotes of T. cruzi attached to the superficial layer with a specialized area of the flagellum. The composition of the cuticle was analyzed by cytochemistry to determine constituents relevant for parasite attachment. Intense staining with the fluorochrome Nile red indicated the presence of lipids, and measurements of contact angles formed by test fluids with the rectal wall revealed that the luminal surface is hydrophobic. The mechanism of attachment of T. cruzi was found to be based on a hydrophobic interaction. The flagellates bound to lipids extracted from the cuticle and to saturated hydrocarbons. Chitin, which has been presumed to be the natural binding substrate of T. cruzi, was localized using gold-labeled wheat-germ lectin. Chitin occurred in the procuticle but was absent from the superficial and epicuticular layers and, thus, is not accessible for binding by T. cruzi. In addition, it could not be confirmed that galactose-specific lectins or heparin receptors mediate flagellate attachment to the rectum.

Anti-heart tissue antibodies during experimental infections with pathogenic and non-pathogenic Trypanosoma cruzi isolates in dogs

Dogs infected with a pathogenic Trypanosoma cruzi isolate (Tc-O), or a non-pathogenic isolate (Tc-D) developed low antinuclear antibody titers throughout 240 days of infection. Dogs infected with Tc-O, but not Tc-D, produced anti-heart tissue antibodies (as measured by an ELISA) throughout infection. Antibody levels were highest immediately after the acute stage of infection (30 days post-inoculation—DPI) when myocardiocyte destruction was at its greatest. Antibody levels progressively declined by 100 DPI, and were almost undetectable by 240 DPI when Tc-O infected dogs developed dilated cardiomyopathy. On Western blots, the anti-heart tissue antibodies recognized an antigen with the same molecular weights (65 and 61 kDa M r) recognized by a monoclonal antibody against human β-adrenoceptors. The results suggest that dogs produce antibodies against heart tissue antigens, possibly the β-adrenoceptor, during infection with a pathogenic, but not a non-pathogenic, isolate of T. cruzi.

The action of Trypanosoma cruzi trans-sialidase on glycolipids and glycoproteins.

Addition of sialic acid residues in the human pathogen Trypanosoma cruzi glycoconjugates is mediated by a trans-sialidase and not by a CMP-sialic acid:glycoconjugate sialyltransferase. Incubation of trans-sialidase with N-[galactose-14C]acetyllactosamine and O-linked oligosaccharides, N-linked glycopeptides (both obtained from fetuin) or sialyllactose showed that the last three compounds were donors of sialic acid residues to the first one. Moreover, N- and O-linked oligosaccharides in asialofetuin and asialomucin, respectively, served as acceptors of sialic acid units. Gangliosides GM3, GD1a and GT1b but not GM2, GM1a nor GD1b donated sialic acid units to N-acetyllactos amine when incubated with trans-sialidase. This showed that only sialic acid units bound to terminal galactosyl residues were transferred. GM1a was converted to GD1a, and GD1b to GT1b when incubated with the appropriate donor. The fact that asialo-GM1a was converted to a ganglioside migrating as GD1a on thin-layer chromatography suggested that sialic acid units may be transferred to internal galactosyl residues, although once linked to those residues they can not be further transferred to other glycoconjugates. Sialic acid residues linked alpha 2,3- but not alpha 2,6- or alpha 2,8- were transferred by the trans-sialidase. Methyl beta-galactoside but not methyl alpha-galactoside served as acceptor of sialic acid units, thus suggesting that terminal alpha-linked galactosyl units in T. cruzi and mammalian glycoproteins are not sialylated by the enzyme. As the trans-sialidase employed in these experiments has been shown to be located on the external surface of the parasite and to be shed to the medium, the relatively broad specificity shown by the enzyme with respect to protein- and lipid-linked oligosaccharides strongly suggests that infection by T. cruzi might alter the sialic acid distribution in glycoproteins and glycolipids of the mammalian host.

Separation and quantitation of the polyamine biosynthesis inhibitor d,l-α-difluoromethylarginine and other guanidine-containing compounds by high-performance liquid chromatography

The arginine decarboxylase inhibitor difluoromethylarginine (DFMA) is an important tool in the study of polyamine metabolism, particularly with respect to the human pathogen Trypanosoma cruzi. This paper demonstrates a unique method for the detection and quantitation of intracellular DFMA using the fluorogenic agent 9,10-phenanthrenequinone. After separation of cell extracts by HPLC, DFMA can be accurately and reproducibly quantified with a lower sensitivity limit of 0.1 nmol by this simple fluorometric method. This assay can also be used to detect other guanidine-containing compounds such as arginine, agmatine, creatinine, and hirudonine, but not substituted guanidines such as aminoguanidine and creatine, or the structurally related amidines such as benzamidine and pentamidine.